Producing ethanol and other fermentation products from biomass has proven to be challenging on multiple fronts. Biomass is inherently recalcitrant to enzymatic and microbial attack, which necessitates a pretreatment to make the crystalline cellulose in the lignocellulosic substrate more available for enzymatic hydrolysis. To complicate the matter further, biomass intrinsically contains microbial inhibitors that are released during pretreatment and enzymatic hydrolysis, affecting fermentation performance. Inhibition can be a function of disruption of cellular replication, disruption of sugar metabolism, or disruption of membrane integrity. There are several classes of fermentation inhibitors encountered in biomass conversion: furfural and hydroxymethyl furfural (furans), phenolic compounds, and weak acids.
When pretreatment conditions are too severe, furfural and 5-HMF are produced from pentose and hexose sugars, respectively. They can be metabolized by yeast to their furfuryl alcohol forms. At low concentrations, furfural can be beneficial to fermentation since yeast can use it to regenerate NAD+, thus reducing glycerol production. However, cell replication is inhibited by furfural and 5-HMF at higher concentrations, inducing an increased lag phase in growth as well as halting anaerobic growth. Ethanol production is also negatively affected at high concentrations. These inhibitors can be avoided to a large extent by optimizing pretreatment conditions for each feedstock.
Weak acids such as acetic acid originate from the de-acetylation of hemicellulose in the biomass or from sugar and lignin degradation during pretreatment (e.g., levulinic acid and formic acid). At fermentation pH (e.g., 5.5), these acids are relatively near their pKa, existing equally in their associated and dissociated states. There are multiple theories as to how these acids inhibit fermentation, such as uncoupling of metabolism or intracellular anionic accumulation. They result in a significant drop in intracellular pH and an inability to produce enough ATP for the organism to survive. Also, since undissociated weak acids can pass through the cell membrane and dissociate in the cytosol, the acid can then interfere with the enzymes used for sugar metabolism. It is inherently difficult to prevent the formation of weak acids—especially acetic acid—since they are intrinsic to the biomass feedstock.
Acetic acid is a known inhibitor of microorganism activity during fermentation. The general practice is to dilute the fermenter/propagator feed stream in order to reduce the concentration of acetic acid below a certain level which allows an acceptable level of microorganism activity. However by doing so, sugars are also diluted and consequently the product concentration is dilute.
Phenolic compounds, like vanillin, syringaldehyde, and ferulate, are a major constituent of lignin and are also linked to hemicellulose in some biomass substrates. These compounds are able to embed themselves into the cell membrane of organisms, causing a loss of integrity. Lower-molecular-weight phenolic acids behave in the same way as weak acids with respect to disruption of intracellular pH.
Because fermentation inhibitors are inevitably produced and present in biomass hydrolysates, what is needed is a practical approach to removing fermentation inhibitors prior to fermentation of sugars derived from any type of biomass (e.g., wood, bagasse, straw, etc.).